Cell signaling 3

1. Cell signaling 3

2. Gaseous signals: NO • Nitric Oxide (NO): major paracrine signaling agent in the nervous system and circulatory system. • NO has a short half-life (about 0.5 sec) and thus can only act over short distances • Major effect is to mediate relaxation of smooth muscle – originally called endothelium-derived relaxing factor. • Pharmacologically, nitroglycerine, a drug for angina, is converted to NO, which dilates systemic arterioles and thus lowers systemic arterial blood pressure and increases coronary blood flow.

3. Generalized Effector Pathway for gaseous messengers NO and CO Inputs to source tissue NO synthetase Arginine + O2 Citrulline + NO Diffusion to target tissue Soluble guanylate cyclase GTP cGMP Protein Kinase G Effects on target cells cGMP phosphodiesterase GMP Heme oxygenase CO inactive

4. “endothelium-derived relaxing factor” is NO

5. Drug targets • Both nitric oxide synthases and phosphodiesterases exist in multiple forms that are tissue-specific

6. A specific example – genital vasculature and sildenafil Acetylcholine – from PS postganglionic cells binds to muscarinic receptors on endothelial cells NO synthetase Arginine + O2 Citrulline + NO Diffusion to corpora cavernosa sm muscle Zowie! Soluble guanylate cyclase Sildenafil, etc GTP cGMP Protein Kinase G Vasodilation= erection Tissue specific phosphodiesterase PDE5 GMP Dietary arginine supplementation may lower blood pressure and improve sexual response

7. Histamine: sources • Basophils - class of leucocytes • Mast cells – scattered throughout tissues • Enterochromaffin-like cells - in stomach

8. Basophil or mast cell -mediates responses to allergens and infectious agents NO, adenosine

9. Histamine is a final common pathway that integrates the effects of neural and hormonal inputs on gastric acid secretion

10. Eicosanoids • 20-C unsaturated fatty acid derivatives • Phospholipase A acts on membrane phospholipids to produce arachidonic acid • Tissue-specific lipoxygenases and synthases then generate various prostaglandins, thromboxanes, leucotrienes, endocannabinoids and isoecosanoids • All receptors for this class of signals are G-protein coupled.

12. Arachidonic acid can be generated directly by activation of Phospholipase A, or indirectly through the generation of DAG and subsequent formation of arachidonic acid from the DAG.

13. Analgesics and antiinflammatory drugs (NSAIDS) target eicosanoids • Aspirin and many other NSAIDs inhibit COX-1, a constitutively expressed enzyme that produces prostacyclins and thromboxanes– responsible for its effects on blood clotting, gastric secretion and pain perception. • To some extent, aspirin and other NSAIDs also inhibit COX-2: expression of COX-2 is induced by inflammation. • COX-2 (cyclooxygenase) inhibitors – designed to be highly specific therapy for rheumatoid arthritis and other inflammatory disease– but specificity was not good enough to eliminate side-effects on clot formation and other physiological processes.

14. Ca++ as a 2nd messenger • Cytoplasmic [Ca++] is tightly controlled at 10-7M or lower. • Rise in cytoplasmic [Ca++] can be the result of Ca++ channel activation, or release from internal stores (ER, mitochondria), or both at the same time. • Effects: • vesicle fusion with plasma membrane in regulated secretion • synaptic vesicle release • muscle contraction

15. Calcium-Calmodulin Signaling • Calcium-binding proteins like calmodulin have a dual function of buffering intracellular Ca++ and transducing its effects.

16. The Ca++/calmodulin complex can activate three major enzyme families • CaM kinases (all cells) - for example, glycogen phosphorylase kinase is a heterotetramer- troponin, a control protein that switches on striated muscle contraction, is a trimer. In both molecules one of the subunits is calmodulin. • Myosin light chain kinases (MLCK) (cardiac and smooth muscle cells – we will see a specific example of this in the smooth muscle lectures) • Protein phosphatase B (calcineurin) (most excitable cell types)

17. Steroid hormones and thyroid hormones • act through cytoplasmic or nuclear receptors • Main mode of action is to influence gene expression

18. Domains of the steroid receptor are specialized for particular functions • Attachment to cytoplasmic heat shock proteins • Interaction with steroid • Transition through nuclear pores • Attachment to DNA • Interaction with transcription control factors

19. This cartoon shows a generalized picture of how steroid receptors work; but there are some variations as shown in the next slide

20. “Glucocorticoid” refers mainly to cortisol, the main hormone of the adrenal cortex, which is involved in modulating response to stress. The unoccupied receptor is cytoplasmic; the complex of hormone and receptor is translocated to the nucleus, where it modulates transcription of specific genes. In contrast, the estrogen receptor, although very similar to the glucocorticoid receptor, is confined to the nucleus. Thyroid hormones (T4 tetraiodothyronine or thyroxine; T3 triiodothyronine – these are tyrosine derivatives and aren’t steroids). Here, the receptor is already bound to the regulatory region of responsive genes and hormone binding causes a reporesor protein to be replaced by a coactivator.

21. All of the major 2nd messenger systems we’ve seen so far, and some we haven’t seen yet, have the potential to affect gene expression – not just the ones for steroids and thyroid hormones.Clearly, we need to know something about control of gene expression. Chapter 5 in Goodman, starting about p 178, may be helpful.

22. What is a gene? • Confers a particular trait? • Piece of DNA that begins with a start signal and sequence and ends with a stop signal? • Codes for a particular protein (or at least, a functional RNA)? • When I say a gene is expressed, I mean that a particular protein is synthesized.

23. Gene expression can be regulated at multiple steps. The one we really care about now is 2. You already know about 1 (think of Barr bodies), and 5, 6 and 7.

24. General picture of transcription control in eucaryotes At the top, the structure of the chromatin is condensed, and those genes cannot be expressed. Cells can only express those portions of the DNA that have been somewhat unwound. The tan discs are histones, which control the metastructure of the chromatin.

25. Steroid and thyroid receptors (and mediators in the other systems) are activators of transcription In order to do its thing, an activator must bind to an upstream region of the DNA called a response element, and then bend around to interact with the RNA polymerase at the start site to start transcription. Repressors can interfere with this effect.

26. It takes two to tango - steroid and thyroid hormone receptors gotta dimerize For some of these receptors, the dimers are homodimers, for others, the receptor must pair with a retinoic acid receptor – if you are a thyroid receptor or vitamin D receptor ya don’t have to dance with the one that brung ya.

27. Now we can look at how the cAMP 2nd messenger system affects gene expression. You already saw how the cytoplasmic elements of this pathway worked in the last lecture. When activated PKA enters the nucleus, it phosphorylates CREB proteins that are associated with the cAMP response element CRE. A nuclear protein called CBP can then bind to the CREBs and activate RNA polymerase to start transcription.

28. And, we can see how receptor tyrosine kinases affect gene expression- again, you’ve seen the first steps of this already.

29. Don’t miss the forest for the trees… • This is a complex pathway, but - • Forget about SOS • Ras is like a G protein alpha subunit– you know about them already • Ras and Raf are like Beavis and Butthead – they just always seem to go together. • MEK (“MAP kinase”) - is just a message-carrier for Ras and Raf • MAPK (stands for mitogen-activated protein kinase) is the one that really does the heavy lifting at the end of this cascade – it mediates both the cytoplasmic effects and the nuclear effects. In the nucleus, it phosphorylates at least 6 known transcription factors, some of which act on multiple genes

30. In the next installment of this series, we will look at some specific examples of hormones at work.